1. Field of the Invention
The present invention relates generally to signaling of TGF-b superfamily. More specifically, the present invention relates to antagonism of signaling of TGF-b superfamily ligands.
2. Description of the Related Art
The transforming growth factor b (TGF-b) superfamily comprise over 30 secreted ligands in human that control cell growth, homeostasis, differentiation, tissue development, immune responses, angiogenesis, wound repair, endocrine function and many other physiologic processes. Members of this superfamily include TGF-b, activins, bone morphogenetic protein (BMP), Growth and Differentiation Factor (GDF) and nodal-related families. Disruption or dysregulation of activin and TGF-b signaling is associated with multiple pathological states including carcinogenesis.
TGF-b superfamily members share a distinct structural framework known as the cystine knot scaffold. Activin and TGF-b are each disulfide-linked dimmers. Activin consists of two b chains. Although there are several activin b subunit genes and an extensive array of possible b-b dimers, only bA-bA (activin-A), bA-bB (activin-AB) and bB-bB (activin-B) have been isolated as dimeric proteins and shown to be biologically active. Three TGF-b genes exist in mammals giving rise to the TGF-b1, TGF-b2 and TGF-b3 isoforms.
Activin and TGF-b Signaling Via Receptor Serine Kinases
TGF-bs, activins and other members of the TGF-b superfamily exert their biological effects by interacting with two types of transmembrane receptors (type I and type II) with intrinsic serine/threonine kinase activities, called receptor serine kinases (RSKs). Type I receptor serine kinases are referred to as ALK1 to 7, for Activin receptor-Like Kinases. The receptor activation mechanism was first established for TGF-b which was shown to bind its type II receptor (TbRII) leading to the recruitment, phosphorylation and activation of its type I receptor (ALK5). A similar mechanism of ligand-mediated receptor assembly and type I receptor phosphorylation has been demonstrated for activin receptors involving initial binding of activin to ActRII or ActRIIB followed by recruitment, phosphorylation and activation of the type I receptor ALK4.
The ligand binding properties of the receptor extracellular domains (ECDs) have been extensively examined. The crystal structure of the ActRII-ECD provided detailed information regarding sites predicted to be involved in receptor:ligand interactions. The crystal structure of the ActRII-ECD bound to BMP-7 has recently been solved and it was shown that the amino acids on ActRII required for activin-A binding make up interfacial contacts between ActRII and BMP-7 and are required for BMP-7 binding. An allosteric conformational change was observed in BMP-7 in its predicted type I receptor binding site following binding to ActRII. This suggested a general model for cooperative type I/type II receptor assembly induced by BMPs (or activin) to form a hexameric complex containing the dimeric ligand, two type II receptors and two type I receptors.
The structure of activin-A bound to the ActRIIB-ECD was also solved recently and was generally consistent with previous findings regarding the activin-A binding site on the ActRIIA receptor. Using the crystal structure of BMP2 bound to the BMP type I receptor (ALK3-ECD) as a guide, an activin-A binding surface on the type I receptor ALK4-ECD was recently identified.
The structure of TGF-b3 bound to the TbRII-ECD has also been solved and indicated unexpectedly that the TGF-b binding interface with its type II receptor is very different from the corresponding interface of activin and BMP7 with ActRII. This suggests that although activin and TGF-b have a similar mechanism of receptor activation, they apparently have unrelated ligand-type II receptor interfaces.
Regardless of the precise mechanism of receptor assembly by TGF-b superfamily ligands, it has been generally established that following receptor assembly, type II receptors phosphorylate type I receptors within a juxtamembrane cytoplasmic glycine- and serine-rich region called the GS domain and this phosphorylation event activates the type I receptor kinase to initiate downstream signaling.
Regulation of Activin and TGF-b Receptor Access
Activins are secreted in their processed, biologically active form. However, the ability of activins to access and assemble signaling receptors can be inhibited in several distinct ways. Inhibins (a-b) share a b subunit with activins and are TGF-b superfamily members that act in conjunction with the membrane proteoglycan betaglycan to form high affinity complexes with activin type II receptors, thereby preventing these receptors from binding activin and initiating signaling. The soluble, extracellular activin binding follistatins bind activins with high-affinity and also block the ability of activin to bind its cell-surface receptors and initiate signaling. In addition, the pseudo (decoy) type I receptor BAMBI (BMP and Activin Membrane-Bound Inhibitor) can bind BMP or activin in non-functional complexes with activin and BMP receptors to block signaling.
Unlike activin, TGF-b isoforms are not secreted in an active form but rather are secreted as inactive “latent” complexes. These complexes comprise the inactive TGF-b dimer in non-covalent complexes with two prosegments to which one of several “latent TGF-b binding proteins” is often linked. Latent TGF-b complexes and their binding proteins associate with the extracellular matrix and await one of several possible activating stimuli to provide a rapidly available pool of releasable TGF-b that can respond to highly localized signals.
Smad Signaling
Based upon genetic studies in Drosophila and Caenorhabditis elegans, a group of proteins now called Smads have been found to transduce signals from receptor serine kinases and mediate regulation of target gene transcription by activin, TGF-b and other TGF-b superfamily members. Structural and functional considerations allow subdivision of Smads into three subfamilies: pathway-specific, common mediator, and inhibitory Smads.
Ligand/receptor assembly and activin receptor-like kinase (ALK) phosphorylation triggers a transient ALK/pathway-specific Smad association during which the ALK phosphorylates the Smad on its last two serine residues in the C terminal SSXS motif. Activin and TGF-b signals are mediated by the pathway-specific Smads, Smad2 and Smad3 and these Smads are sequestered near their signaling receptors by Smad Anchor for Receptor Activation (SARA), a cytoplasmic membrane-associated protein that has been shown to facilitate Smad2/3 signaling.
Once activated, Smad2 and Smad3 form hetero-oligomeric complexes with the common mediator Smad, Smad4, that was first discovered in humans as the pancreatic tumor suppressor gene, DPC4. Smad2/3/4 complexes translocate to the nucleus and interact directly with DNA and/or with cell-type specific co-activator or co-repressor proteins leading to the activation or repression of target genes.
Two vertebrate inhibitory Smads have been identified, Smad6 and 7, which lack the C-terminal SSXS motif found in the pathway specific Smads. Smad6 and 7 are inhibitors of Smad signaling and bind to activin receptor-like kinases (ALKs) to prevent phosphorylation and activation of the pathway-specific Smads. In transfected cells, Smad7 inhibits transcriptional responses induced by activin or TFG-b or by a constitutively active ALK4. Smad7 may therefore provide an intracellular feedback signal to restrain the effects of activin and TFG-b.
Smad2/3 Signaling and Growth Control
TGF-b and activin are both well known for their ability to inhibit proliferation of multiple cell types including most epithelial cells, and gene expression profiling has indicated essential similarity of transcriptional responses to constitutively active activin or TGF-b type I receptors in cancer cells. Activation of the Smad2/3 signaling pathway leads to inhibition of cell cycle progression during G1 and in some cases terminal differentiation, or apoptosis. The growth inhibitory response to Smad2/3 signals has been divided into two major classes: gene responses that lead to inhibition of cyclin-dependent kinases (cdks) and down regulation of c-myc.
The retinoblastoma tumor suppressor protein (pRb) and its family members p107 and p130 control cell cycle progression and have activity that is regulated by cdk phosphorylation. TGF-b signals have been shown to induce cdk inhibitors including p15INK4B (p15) and p21CIP1/WAF1 (p21) and to down regulate the tyrosine phosphatase cdc25A. p15 binds and inactivates cdk4 and cdk6 causing displacement of p27 from cyclin D-cdk4/6, allowing it to bind and inhibit cyclin E-cdk2. p21 also binds and inhibits cyclin E-cdk2. cdc25A is an activator of cyclin D-cdk4 and its down regulation therefore reduces the activity of this cdk. Overall, decreased cdk activity in response to Smad2/3 signaling reduces pRb phosphorylation by these cdks, allowing pRb to prevent E2F function and block cell cycle progression.
Unlike cdk inhibition, which exhibits cell type dependent diversity, down regulation of c-Myc, a member of the basic helix-loop-helix leucine zipper (bHLH-LZ) family of transcription factors, is observed in most cell types that are growth inhibited by Smad2/3 signals. In addition, down regulation of c-Myc by Smad signals is required for the inactivation of cdks, and evidence also implicates c-Myc as a positive regulator of cdc25A expression. It was recently shown that E2F4/5 proteins and the Rb protein p107 form a pre-formed complex with Smad3 in the cytoplasm that awaits TGF-b receptor activation, Smad3 phosphorylation and Smad4 assembly leading to translocation of the complex to the nucleus to bind the c-myc promoter and repression of the c-myc gene.
The Id family of transcriptional regulators inhibit terminal differentiation, promote cell proliferation and have been implicated in cancer. Myc and Id proteins can form complexes that cooperate to override the tumor suppressor function of pRb. Interestingly, it was recently shown that TGF-b causes repression of Id gene expression via preassembled, cytoplasmic Smad3-ATF3 complexes that translocate to the nucleus with Smad4 and target Id promoters following TGF-b receptor activation. It was also recently demonstrated that key cellular responses to TGF-b signals, including induction of the cdk inhibitor p21, rely on direct interactions between Smad2 and the tumor suppressor and transcriptional regulator p53. In summary, these results indicate that Smad2 and Smad3 likely play essential but distinct roles in regulating cell proliferation.
Smad2/3 Pathway and Cancer
It is not surprising that disruptions or alterations in the activin and TGF-b signaling pathways have been observed in several types of human cancer. Inactivating mutations in TbRII have been observed in colorectal and gastric carcinomas and inactivation of ActRII was recently observed in gastrointestinal cancers. An inactivating mutation in TbRI (ALK5) occurs in one third of ovarian cancers observed and ALK4 mutations have been described in pancreatic cancer leading to the designation of ALK4 as a tumor suppressor gene.
The activin/TGF-b signaling pathway is also disrupted by mutations in Smad4 and Smad2. As mentioned above, Smad4 was originally identified as DPC4 (deleted in pancreatic carcinoma locus 4) and this gene is functionally absent in half of all pancreatic cancers and one third of colon carcinomas. Smad2 is also inactivated in a small proportion of colorectal cancers and lung cancers. Although Smad3 mutations have not yet been observed in human cancers, Smad3−/− mice developed colorectal cancer.
Interestingly, despite its antiproliferative effects, Smad2/3 signaling can also exacerbate the cancer phenotype under conditions in which cells have become refractory to Smad2/3-induced growth inhibition. For example, increased production of TGF-b or activin by tumor cells that are no longer growth inhibited by Smad2/3 signals may lead to increased angiogenesis, decreased immune surveillance and/or an increase in the epithelial to mesenchymal transition (EMT) of tumor cells. Collectively, these effects can lead to increased tumor growth and metastasis.
Epidermal Growth Factor-Cripto, FRL-1, Cryptic (EGF-CFC) Protein Family
Similar to activin, members of the nodal family and GDF-1/Vg1 have been shown to signal via the activin receptors ActRII/IIB and ALK4. Unlike activin, however, these TGF-b superfamily members require additional co-receptors from the Epidermal Growth Factor-Cripto, FRL-1, Cryptic (EGF-CFC) protein family to assemble type II and type I receptors and generate signals.
The EGF-CFC family consists of small, glycosylated, extracellular signaling proteins including human and mouse Cripto and Cryptic, Xenopus FRL-1 and zebrafish one-eyed pinhead (oep). EGF-CFC proteins are known to act as anchored cell surface co-receptors but they also have activity when expressed as soluble proteins or when they are secreted from the cell surface following enzymatic cleavage of their GPI anchor. Genetic studies in zebrafish and mice have shown that EGF-CFC proteins are required for mesoderm and endoderm formation, cardiogenesis, and the establishment of left/right asymmetry during embryonic development. Cripto knockout mouse embryos lack a primitive streak and fail to form embryonic mesoderm. This phenotype is very similar to that observed in ActRIIA−/−; ActRIIB−/− mice, ALK4−/− mice and Nodal−/− mice, consistent with a requirement for nodal signaling via activin receptors and a role for Cripto to initiate primitive streak elongation and mesoderm formation.
It has been shown that Cripto independently binds nodal via its EGF-like domain and ALK4 via its CFC domain. Furthermore, selected point mutations in Cripto that block nodal binding or ALK4 binding disrupt nodal signaling. Substantial biochemical evidence indicates that nodal and Vg1/GDF1 form a complex with activin receptors only in the presence of EGF-CFC proteins.
Cripto is a Tumor Growth Factor
Cripto is an EGF-CFC protein that was first isolated as a putative oncogene from a human teratocarcinoma cell line and it was subsequently shown to be able to confer anchorage independent growth to NOG-8 mouse mammary epithelial cells. Cripto is expressed at high levels in human breast, colon, stomach, pancreas, lung, ovary, endometrial, testis, bladder and prostate tumors while being absent or expressed at low levels in their normal counterparts. The elucidation of the signals and transcriptional events underlying the high level of Cripto expression in these tumors remains an important area of future research.
With regard to Cripto's mechanism(s) of mitogenic action, it has been shown that recombinant, soluble Cripto and a synthetic 47 amino acid Cripto fragment spanning the EGF-like domain can activate both the mitogen activated protein kinase (MAPK) pathway and the phosphatidylinositol-3-kinase (PI3K) pathway. Treatment of HC-11 mammary epithelial cells with soluble Cripto or the 47-mer peptide resulted in tyrosine phosphorylation of the SH2-adaptor protein Shc, association of Shc with Grb2 and activation of the p42/44 Erk/MAPK pathway. It was also shown that soluble Cripto caused phosphorylation of the p85 regulatory subunit of PI3K leading to phosphorylation and activation of AKT in SiHa cervical carcinoma cells. Cripto does not bind to members of the EGF receptor family, although [125I]-Cripto specifically labeled breast cancer cell lines and formed crosslinked complexes with 60 kDa and 130 kDa membrane proteins. Although these proteins were not identified, the 60 kDa protein may have been ALK4.
It was recently shown that the cytoplasmic tyrosine kinase c-Src can be activated by soluble Cripto and that its activity is required for activation of the MAPK/PI3K pathways by Cripto. The GPI-anchored proteoglycan glypican was also reported to be important in facilitating these Cripto signals and glypican was also shown to bind Cripto in a manner dependent on glycanation of glypican. The ability of Cripto to activate the MAPK and PI3K pathways, which are frequently growth-stimulatory in nature, has generally been proposed to explain Cripto's oncogenic effects.
Smad Signaling, Cripto and Cancer
The first demonstration of a physiologic role for TGF-b was its potent and reversible inhibition of developing mouse mammary gland in situ. TGF-b is now well established as an important inhibitor of mammary ductal growth and branching in vivo and over 90% of mammary carcinomas are ductal in nature. Loss of TbRII has been associated with increased risk of invasive breast cancer in women. Consistent with a role in regulating mammary ductal growth, TGF-b1 heterozygous null mice display accelerated mammary epithelial proliferation and ductal outgrowth. Furthermore, transgenic expression of a dominant negative TbRII construct in mammary gland diminishes responsiveness to TGF-b and caused increased incidence of tumors in response to carcinogen relative to control mice. Conversely, transgenic overexpression of TGF-b1 in mammary gland protects against chemical-induced tumors. These results provide direct evidence that TGF-b signaling can actively prevent tumorigenesis in mouse mammary gland. There is also evidence that activin inhibits proliferation of both primary and transformed mammary epithelial cells. Together, these results indicate the importance of the Smad2/3 pathway in inhibiting mammary epithelial cell proliferation and tumorigenesis.
Cripto is overexpressed in many types of human tumors, including ˜80% of breast carcinomas, while its expression is low or absent in their normal counterparts. In contrast to TGF-b, Cripto promotes growth in mammary cells and Cripto overexpression transforms mouse NOG-8 and CID-9 mammary epithelial cells. Cripto overexpression in these cell lines enabled them to grow in soft agar and each displayed an enhanced proliferation rate in monolayer culture. These cells were, however, unable to form tumors in nude mice.
It was also shown that targeted disruption of endogenous Cripto in CID-9 cells via a retroviral antisense construct led to a decreased rate of cellular proliferation. Both the soluble Cripto protein and the 47 amino acid EGF-like domain Cripto peptide have also been shown to facilitate ductal branching and cause mammary ductal hyperplasia. As discussed above, these effects have been explained as the result of the ability of Cripto to activate mitogenic signaling pathways including the MAPK and PI3K pathways. However, many of the growth-related effects of Cripto are also generally consistent with antagonism of the Smad2/3 pathway.
The prior art is lacking in evidence on whether Cripto can play a dual role as an oncogene, not only acts by activating mitogenic MAPK/PI3K pathways, but also antagonizes the antiproliferative Smad2/3 pathway. The present invention thus studies the oncogenic mechanism of Cripto protein in order to gain insight into its effects on activin/TGF-b signaling.